Blood disorders are medical conditions which affect one or more components of the blood, including red blood cells, white blood cells and platelets.
For example, hemoglobinopathies are a group of inherited blood disorders involving defective hemoglobin (Hb) production. The different hemoglobinopathies fall into two main groups: thalassemia syndromes, characterized by reduced synthesis of globin subunits that are structurally normal, and structural hemoglobin variants (abnormal hemoglobins) syndromes, characterized by the synthesis of structurally abnormal globin subunits. Both groups are caused by mutations and/or deletions in the globin related genes. Some hemoglobinopathies are mixed forms that combine features of the two main groups. The most common clinical manifestations of hemoglobinopathies include anemia of variable severity and variable pathophysiology.
In the thalassemia syndromes, which are autosomal recessive conditions, the decrease in globin chain production may result from gene deletion or mutations that adversely affect the transcription or stability of mRNA products. The main types of thalassemia are α-thalassemia, which results from a deletion or mutation in one or more of the four α-globin genes, and β-thalassemia, which results from a deletion or mutation in one or more of the two β-globin genes.
The main two types of thalassemia are further classified based on their severity and the number of gene copies that are affected. β-thalassemia includes β-thalassemia minor (also called (β-thalassemia trait), intermedia and major (also called Cooley's anemia). Generally, if one β-globin gene is affected, thalassemia minor is the result. If the two (β-globin genes are affected, the result is thalassemia intermedia or major, depending on how severely the gene is affected. The genes can be mutated or even completely deleted.
α-thalassemia includes a silent carrier state, where one gene is affected, α-thalassemia trait, where two genes are affected, hemoglobin H disease, where three genes are affected, and α-thalassemia major (also called Hb Bart's hydrops fetalis), where four genes are affected. The latter is a sever condition typically diagnosed in utero, and fetuses with α-thalassemia major are usually miscarried, stillborn, or die shortly after birth.
In addition to decreased hemoglobin production, manifestations of the thalassemias are complicated by the resulting chain imbalance: in α-thalassemia, the β- and/or γ-globin chains are produced in excess. Similarly, in β-thalassemia, α-chains are produced in excess. Hemoglobin chain imbalance damages, shortens life span, and sometimes even destroys red blood cells, leading to anemia.
In the structural hemoglobin variants syndromes, which are autosomal dominant conditions, the structural defects of the globin chains result from an altered amino acid sequence in the α or β chains. Hundreds of hemoglobin variants have been hitherto described, the majority of which are clinically benign or negligible. Some, however, are associated with pathology. The clinically significant variants are divided into four groups. The first group includes variants with a tendency to aggregate, which are associated with sickle cell syndromes, e.g. the HbS variant. The point mutation in the β-globin gene that produces HbS exerts its effect by causing precipitation and polymerization of the HbS with resulting sickling of the red cells. These sickled cells lack deformability, occlude the microvasculature, and lead to necrosis and tissue infarctions, which manifest as painful sickle cell crises. The permanently deformed cells are subsequently removed from the circulation well before the usual 120-day life span of a healthy red cell, contributing to a chronic non-hemolytic and/or hemolytic anemia. The clinical manifestations generally occur only in individuals with homozygous sickle cell disease.
The second group of Hb variants includes variants with abnormal hemoglobin synthesis, e.g. HbE. The third group includes variants with a tendency to precipitate, which are associated with hemolysis (fragile red blood cells), e.g. Hb Köln. The fourth group includes variants with abnormal oxygen transportation and congenital polycythemia, e.g. Hb Johnstown, or with congenital cyanosis (abnormal methemoglobins, HbM abnormalities, e.g. M-Iwate).
Treatment of the different hemoglobinopathies depends on the severity of the disease. For example, hematopoietic stem-cell transplantation is the preferred treatment for the severe forms of thalassemia. Supportive, rather than curative, treatment includes periodic blood transfusions for life, combined with iron chelation. Drugs to treat the symptoms of sickle-cell disease include analgesics, antibiotics, corticosteroids, ACE inhibitors and hydroxyurea.
A growing body of experimental and clinical evidence point to the important role played by oxidative stress in hemoglobinopathies. For example, it has been shown previously that blood cells derived from patients with β-thalassemia and sickle cell anemia, including red blood cells (RBC), platelets and polymorphonuclear neutrophils (PMN), are under oxidative stress—their reactive oxygen species (ROS) were higher than normal and their reduced glutathione (GSH) level was lower than normal. Oxidative stress in thalassemia is thought to be caused primarily by the RBC abnormalities, namely, degradation of unstable Hb which results in free globin chains and heme. Another contributing factor is iron overload due to increased intestinal absorption and therapeutic blood transfusions. Several studies indicated the presence of elevated levels of free radicals, as well as iron-containing compounds, probably released from damaged RBC, in thalassemic plasma.
Oxidative stress in hemoglobinopathies may explain clinical symptoms, for example, the anemia due to the short survival of mature RBC in the circulation. The oxidative stress of platelets could account for their increased tendency to undergo activation and aggregation and the high incidence of thromboembolic complications in these patients. The chronic oxidative stress of the PMN may result in ineffective bactericidal activity which may cause recurrent infections.
An additional blood disorder known to be associated with oxidative stress is hemolytic uremic syndrome (HUS). HUS is a condition that results from an abnormal premature destruction of red blood cells. It often occurs following a gastrointestinal infection with Escherichia coli, which produces toxic substances that destroy the cells. The condition has also been linked to other gastrointestinal infections, including Shigella and Salmonella, as well as non-gastrointestinal infections and other factors such as adverse drug reaction and drug overdose. The damaged red blood cells usually clog the filtering system in the kidneys, which may eventually cause a life-threatening kidney failure. Treatment of HUS may include fluid replacement, RBC transfusions, platelet transfusions, plasma exchange, kidney dialysis, and medications such as corticosteroids.
HUS was found to involve changes in RBCs lipid peroxidation, reduced glutathione (GSH) levels and impaired metabolism, reduced antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase) and abnormal hemoglobin metabolites' levels. It has been suggested that in the acute phase of HUS, RBCs are exposed to an oxidative stress that could contribute to hemolysis directly through oxidative damage and/or by membrane permeability and impaired membrane fluidity.
Another disorder known to be associated with reduced GSH levels, oxidative stress and destruction of red blood cells is glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is an X-linked recessive hereditary disease characterized by abnormally low levels of the enzyme glucose-6-phosphate dehydrogenase. This enzyme is a metabolic enzyme involved in the pentose phosphate pathway, which is particularly important in red blood cell metabolism. G6PD also plays an important role in the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is necessary for the regeneration of reduced glutathione through reducing oxidized glutathione (GSSG). G6PD deficient red blood cells are therefore highly vulnerable to oxidative damage and tend to hemolyze.
The most frequent clinical manifestations of G6PD deficiency include neonatal jaundice, acute hemolytic anemia and favism, which is usually triggered by exogenous agents e.g. toxins, oxidants and certain food, most notably fava beans. Some G6PD variants cause chronic hemolysis. The most effective management of G6PD deficiency is avoidance of drugs or substances known to initiate hemolysis in G6PD deficiency patients.
Thus, effective compositions and methods for treating blood disorders associated with decreased levels of reduced glutathione (GSH) and/or oxidative stress, are needed.
Another exemplary condition where oxidative stress is thought to play a role and contribute to destruction or damaging of cells is preservation of biological samples.
For example, blood samples obtained from individuals are typically processed and stored until needed for use (transfusions and/or fractionation). Either whole blood or processed blood products, including for example red blood cell concentrates (packed red blood cells) and platelet concentrates, are stored under conditions aimed at preserving optimal viability and functioning during the maximum allowed storage period. For example, red blood cells are typically stored in a citrate-phosphate-dextrose (CPD) and/or saline-adenine-glucose-mannitol (SAGM) solution, at 2-8° C. Platelets are typically stored at 20-24° C. with sufficient non-stop agitation to permit good oxygenation and prevent aggregation.
In general, additive solutions for preservation of blood contain anticoagulants, sugars as an energy source, inorganic salts as agents for adjusting pH and osmotic pressure, and adenine as an agent for preventing consumption of blood ATP (adenosine triphosphate), ADP (adenosine diphosphate) and AMP (adenosine monophosphate). In addition to CPD and SAGM noted above, other known additive solutions include mannitol-adenine-phosphate (MAP) and phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM).
However, even under current optimal storage conditions, modifications and/or degradation of blood components occur in the samples. These alterations, known as “storage lesions”, affect lifespan and quality of the stored blood products.
Red blood cells, for example, undergo biochemical changes, such as reduction in the levels of GSH, ATP, which is necessary for multiple cellular processes, and 2,3-diphosphoglycerat (2,3-DPG), which is important for oxygen release. Biomechanical changes also occur, such as altered shape, deformability, aggregability, flexibility and intracellular viscosity. Protein modifications resulting from oxidative stress and progressive hemolysis are also observed. The storage period of red blood cells is currently limited for about 5-7 weeks under optimal conditions. Platelet storage lesions typically include reduction in the levels of GSH, morphological changes, platelet activation and platelet proteolysis. Their storage period is currently limited for up to 5 days under optimal conditions.
Improved compositions and methods for storing biological material, such as red blood cells, for extended periods of time, while maintaining their functionality and viability are needed.
Thiol (—SH) containing compounds are a type of molecules capable of neutralizing several types of damaging oxidative species, thus acting as reducing reagents. The activity of this group of compounds is mainly due to the sulfur atom they comprise which participates in nucleophilic attack on toxic electrophiles, scavenging free radicals, effecting repair of damaged targets through hydrogen atom donation, altering the redox status of the cell, or affecting gene transcription or protein function.
Thiol containing compounds include natural molecules, produced by all living organisms including animals and plants, as well as synthetic molecules. Examples of natural thiol containing antioxidants include reduced glutathione, which is one of the most potent and important antioxidants in mammals, thioredoxins and cysteine.
Examples of synthetic thiol containing redox molecules include N-acetylcysteine amide. Grinberg et al. (2005) Free Radic Biol Med, 38(1); 136-145 tested N-acetylcysteine amide for its antioxidant and protective effects using human red blood cells as a model.
Amer et al. (2008) Biochim. Biophys. Acta, 1780(2):249-55 describe in vitro and in vivo effects of N-acetylcysteine amide as an antioxidant in blood cells of β-thalassemic mice and patients
WO 2002/034202 discloses an antioxidant compound characterized by (a) a peptide including at least three amino acid residues of which at least two are cysteine residues, each having a readily oxidizable sulfhydryl group for effecting antioxidation; and at least two peptide bonds, each being cleavable by at least one intracellular peptidase; and (b) a first hydrophobic or non-charged moiety being attached to an amino terminal of the peptide via a first bond and a second hydrophobic or non-charged moiety being attached to a carboxy terminal of the peptide via a second bond, the first hydrophobic or non-charged moiety and the second hydrophobic or non-charged moiety are selected so as to provide the antioxidant compound with membrane miscibility properties for permitting the antioxidant compound to cross cellular membranes; wherein cleavage of the at least two peptide bonds by the at least one intracellular peptidase results in generation of a plurality of antioxidant species, each including one of the cysteine residues having the readily oxidizable sulfhydryl group and which is also active in effecting antioxidation, thereby providing for a plurality of different antioxidant species acting in synergy in exerting antioxidation.
WO 2012/098546, to the inventor of the present invention and others, discloses potent compounds having combined antioxidant, anti-inflammatory, anti-radiation and metal chelating properties. Short peptides having said properties and methods and uses of such short peptides in clinical and cosmetic applications are disclosed. Among other peptides, Cys-Lys-Met-Cys (SEQ ID NO: 1), Cys-Met-Lys-Cys (SEQ ID NO: 2) and Cys-β-Ala-His-Cys (SEQ ID NO: 3) are disclosed.
There still remains a need for more effective and/or safer compositions and methods that may be useful to meet the above described needs.